Isoprenoids or terpenoids represent the largest class of natural products with more than 40,000 known structures. Many of these terpenoids have immense commercial value. Their biosynthesis in heterologous hosts is relatively easy and cost effective as compared to the chemical synthesis or the extraction from their natural sources. Saccharomyces cerevisiae is choice of organism as heterologous host due to its preference by industry. To increase the yield of isoprenoids in yeast, previous efforts were on manipulating the mevalonate pathway. Using known information about the mevalonate-isoprenoid pathway, three potential targets were identified as being potential blocks for isoprenoid biosynthesis, HMG-CoA reductase (3-hydroxy-3-methylglutaryl-Coenzyme A reductase-HMG1), the transcription factor, UPC2, and the ergosterol branch point, squalene synthase ERG9. Using truncated tHMG1 (that lacks feedback regulation), a hyperactive transcription factor sterol regulatory element binding protein (upc2-1) (that increases expression of the mevalonate pathway), or reduced expression of ERG9 (that prevents isoprenoids from branching off), increased flux has been demonstrated and the yield of isoprenoids further increases when these different mutations are combined. However, in the cell, metabolic pathways are interconnected and tightly regulated and it is possible that besides the mevalonate pathway genes, there may be other genes which affect directly or indirectly the yield of carotenoids or other terpenoids produced in yeasts. However, a good genetic screening method is required to identify the same. As carotenoids are colored compounds, their production by yeast cells provides a good visual phenotypic screen. Depending on the level of expression of carotenogenic genes the color imparted varies from faint yellow, yellow to orange. Their color can be used as visual genetic screen for determining flux in the isoprenoid pathway.
Several groups have attempted to increase the flux in the isoprenoid pathway using this carotenoid based visual pigmentation screen using the enzymes from Xanthophyllomyces dendrorhous, however these studies have met with limited success, since in all it was observed that increasing the flux in this pathway through known flux increasers such as tHMG1, a decrease in pigmentation was observed. Estimation of carotenoids revealed that the decrease was most likely due to a block at the phytoene dehydrogenase (RtCRTI) step since phytoene was accumulating under these conditions and the increased phytoene (which is colorless) masked any increase in color due to higher β-carotene and prevented the use of visual pigmentation as a genetic screen.
In one study the transformed yeast deletion collection with the carotenogenic plasmids from Xanthophyllomyces dendrorhous were screened for altered pigmentation, and it was observed that approximately 1100 deletion strains showed a decrease in color while 156 deletion strains showed an increase in color. Among the latter 5 showed a consistent four-fold increase in carotenoids levels as compared to parent strain. However, when attempts were made to validate the increased flux by examining production of bisabolene in these strains, all strains surprisingly produced less bisabolene than wild type parental strain. The slight increase in bisabolene production in these deletion strains that was eventually achieved was only possible after carrying out several other modifications (such as gene fusions) of the isoprenoid pathway. Thus, the visual screen, which was not validated by known flux increasers (such as tHMG1) did not really succeed in picking up the desired phenotypes.
The red yeasts belonging to the Rhodotorula spp., Rhodosporidium spp. and Sporobolomyces spp. have an intense red color and are considered to have highest β-carotene levels. In addition to β-carotene, these yeasts produce the carotenoids torulene and torularhodin. To overcome the limitations encountered in the increased production of isoprenoid by carotenoid based visual pigmentation screen, the present invention aims to use the carotenogenic genes from these red yeasts and attempts to develop a genetic screen for isoprenoid/carotenoid production in Saccharomyces cerevisiae. The carotenoid gene of Arabidopsis spp. and plants is also expressed in yeast. The carotenoids of red yeasts are being expressed in S. cerevisiae for the first time. The invention seeks first to identify and express these enzymes from this yeast followed by a strategy to develop it as a genetic screen for carotenoid/isoprenoid production by overcoming limiting step i.e desaturation of phytoene by phytoene dehydrogenase. The invention aims to optimize genetic screen by utilizing catalytically efficient mutant of phytoene dehydrogenase. The present invention also aims to increase α-farnesene of the isoprenoid pathway. The present invention also provides two mutant genes—phytoene dehydrogenase (RtCRTI) and SPT15 which increase flux in isoprenoid pathway and hence the yield of carotenoids.